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Updated: Jan 30, 2026

Colorimetric Analysis of Alkaline Phosphatase Activity in S. aureus Biofilm
Published on: April 12, 2019
Molecular structure regulation and enzyme cascade signal amplification strategy for upconversion ratiometric
Hongyu Chen1, Zile Zhou1, Qiujun Lu1
1Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education), College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha, 410081, China.
Abstract:
Herein, molecular structure regulation and enzyme cascade signals amplification (MRECAmp) strategy was proposed to construct a novel upconversion ratiometric fluorescence and colorimetric dual-readout assay platform for highly sensitive and selective detection of alkaline phosphatase (ALP) activity. The detection strategy is divided into three aspects. Firstly, Ag+ oxidates o-phenylenediamine (OPD) to 2,3-diaminophennazine (OPDox), which can significantly quench upconversion fluorescence at 487 nm based on inner filter effects (IFE) while the upconversion emission at 668 nm was essentially unchanged for Zn2+-doped NaYF4:Yb3+,Er3+,Tm3+ upconversion nanoparticles (UCNPs) probes under the near-infrared (NIR) irradiation. Secondly, ALP catalyzes the hydrolysis of the substrate l-ascorbic acid 2-phosphate (AAP) to ascorbic acid (AA) which can consume part of Ag+ to inhibit the generation of OPDox and the AA was oxidized to dehydroascorbic acid (DHAA). The specific performance of DHAA molecular structure regulation, due to the condensation reaction between dicarbonyl group of DHAA and diamine group of OPD, that is confirmed by ESI-MS and 1H NMR spectra analysis, can remarkably enhance the selectivity of ALP detection, which is conceptually different from the previously reported ALP fluorescent assays. Thirdly, the generated DHAA reacts with OPD to form 3-(dihydroxyethyl)furo [3,4-b]quinoxaline-1-one (DFQ) that further inhibits the generation of OPDox, which makes the enzyme-controlled cascade signal amplification (ECAmp) can be realized with a great fluorescence recovery (60.04%) at 487 nm. Therefore, the fluorescence response signal ((I487/I668)0/(I487/I668)) is amplified by ECAmp strategy, allowing the quantitative analysis of ALP with a detection limit of 0.03 mU/mL. The detection of ALP activity in human serum samples over the conventional methods demonstrates that the MRECAmp method provides a sensing platform for probing ALP activity and shows promising outlook in biomedical studies.
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